DESCRIPTION (Adapted from applicant's description): Defects in the cell cycle regulatory machinery are commonly found in cancer cells. Recently, the Anaphase Promoting Complex (APC) was identified as a highly conserved complex that regulates mitotic cyclin degradation in Xenopus, human and yeast cells. It now appears that the APC is an important global regulator of the cell cycle in that it targets substrates which are important in 1) the regulation of the entry into anaphase, 2) the exit from mitosis, and 3) the assembly of the mitotic spindle for proteolysis. Current data suggests that the three known substrates for the APC in budding yeast - Pds1, Clb2, and Ase1 - are degraded at different times. These findings point to the existence of specificity determinants for the degradation APC substrates. Because the timing of Ase1 degradation appears to differ from that of the other two budding yeast APC substrates, and because its specific localization allows ASE1 degradation to be followed in individual cells, the candidate has used Ase1 to develop tools to attack the question of APC substrate recognition. The goal of this project is to define the cis-acting and trans-acting factors through which Ase1 is recognized by the APC. The precise timing of degradation of Pds1, Clb2, and Ase1 will be determined through block and release experiments with the three genes under the control of the repressible MET25 promoter. The role of cis-acting signals will be assayed by destruction box (the only cis-acting signal for APC-mediated proteolysis) swap experiments and by site-directed mutagenesis of putative phosphorylation sites in Ase1. Trans-acting factors will be identified by affinity chromatography with the Ase1 degron.